Method of synthesizing polynucleotides using ionic liquids

ABSTRACT

A method of synthesizing polynucleotides is disclosed. The method involves contacting a first nucleotide with a selected reactive group in the presence of an ionic liquid. The selected reactive group may be on a second nucleotide, a polynucleotide, or on a moiety on an insoluble substrate, for example in an oligonucleotide synthesizer.

RELATED APPLICATION

[0001] This application relates to a U.S. patent application entitled“Use of Ionic Liquids for Fabrication of Polynucleotide Arrays”, filedon the same day as this application in the names of Myerson et al.,Agilent docket number 10011388.

DESCRIPTION

[0002] 1. Field of the Invention

[0003] The invention relates generally to methods of polynucleotidesynthesis. The invention more specifically relates to forminginternucleotide bonds in a solution containing ionic liquid.

[0004] 2. Background of the Invention

[0005] Much interest has been focused on reactions for couplingnucleotides to form polynucleotide chains, and various chemical schemeshave been described for the synthesis of polynucleotides. Typicallythese methods use a nucleoside reagent of the formula:

[0006] in which:

[0007] A represents H or an optionally protected hydroxyl group;

[0008] B is a purine or pyrimidine base whose exocyclic amine functionalgroup is optionally protected;

[0009] one of M or Q is a conventional protective group for the 3′ or5′-OH functional group while the other is:

[0010] where x may be 0 or 1, provided that:

[0011] a) when x=1:

[0012] R′ represents H and R″ represents a negatively charged oxygenatom; or

[0013] R′ is an oxygen atom and R″ represents either an oxygen atom oran oxygen atom carrying a protecting group; and

[0014] b) when x=0, R′ is an oxygen atom carrying a protecting group andR″ is either a hydrogen or a di-substituted amine group.

[0015] When x is equal to 1, R′ is an oxygen atom and R″ is an oxygenatom, the method is in this case the so-called phosphodiester method;when R″ is an oxygen atom carrying a protecting group, the method is inthis case the so-called phosphotriester method.

[0016] When x is equal to 1, R′ is a hydrogen atom and R″ is anegatively charged oxygen atom, the method is known as the H-phosphonatemethod.

[0017] When x is equal to 0, R′ is an oxygen atom carrying a protectinggroup and R″ is a halogen, the method is known as the phosphite method,and when R″ is a leaving group of the disubstituted amine type, themethod is known as the phosphoramidite method.

[0018] The conventional sequence used to prepare an oligonucleotideusing reagents of the type of formula (I), basically follows fourseparate steps: (a) coupling a selected nucleoside which also has aprotected hydroxy group, through a phosphite linkage to a functionalizedsupport in the first iteration, or a nucleoside bound to the substrate(i.e. the nucleoside-modified substrate) in subsequent iterations; (b)optionally, but preferably, blocking unreacted hydroxyl groups on thesubstrate bound nucleoside; (c) oxidizing the phosphite linkage of step(a) to form a phosphate linkage; and (d) removing the protecting group(“deprotection”) from the now substrate bound nucleoside coupled in step(a), to generate a reactive site for the next cycle of these steps. Thefunctionalized support (in the first cycle) or deprotected couplednucleoside (in subsequent cycles) provides a substrate bound moiety witha linking group for forming the phosphite linkage with a next nucleosideto be coupled in step (a). Final deprotection of nucleoside bases can beaccomplished using alkaline conditions such as ammonium hydroxide, in aknown manner.

[0019] The foregoing methods of preparing polynucleotides are well knownand described in detail, for example, in Caruthers, Science 230:281-285, 1985; Itakura et al., Ann. Rev. Biochem. 53: 323-356;Hunkapillar et al., Nature 310: 105-110, 1984; and in “Synthesis ofOligonucleotide Derivatives in Design and Targeted Reaction ofOligonucleotide Derivatives, CRC Press, Boca Raton, Fla., pages 100 etseq., U.S. Pat. No. 4,415,732, U.S. Pat. No. 4,458,066, U.S. Pat. No.4,500,707, U.S. Pat. No. 5,153,319, U.S. Pat. No. 5,869,643, EP 0294196,and elsewhere. The phosphoramidite and phosphite triester approaches aremost broadly used, but other approaches include the phosphodiesterapproach, the phosphotriester approach and the H-phosphonate approach.Such approaches are described in Beaucage et al., Tetrahedron (1992)12:2223-2311. A more recent approach for synthesis of polynucleotides isdescribed in U.S. Pat. No. 6,222,030 B1 to Dellinger et al, Issued Apr.24, 2001.

[0020] In the typical phosphoramidite method of solid phaseoligonucleotide synthesis, the synthesis typically proceeds in the 3′ to5′ direction (referring to the sugar component of the added nucleoside),although the synthesis may easily be conducted in the reverse direction.The added nucleoside generally has a dimethoxytrityl protecting group onits 5′ hydroxyl and a phosphoramidite functionality on its 3′ hydroxylposition. Beaucage et al. (1981) Tetrahedron Lett. 22:1859. See FIG. 1for a schematic representation of this technology. In FIG. 1 “B”represents a purine or pyrimidine base, “DMT” represents dimethoxytritylprotecting group and “iPr” represents isopropyl. In the first step ofthe synthesis cycle, the “coupling” step, the 5′ end of the growingchain is coupled with the 3′ phosphoramidite of the incoming monomer toform a phosphite triester intermediate (the 5′ hydroxyl protecting groupprevents more than one monomer per synthesis cycle from attaching to thegrowing chain). Matteucci et al. (1981) J. Am. Chem. Soc. 103:3185.Next, the optional “capping reaction” is used to stop the synthesis onany chains having an unreacted 5′ hydroxyl, which would be onenucleotide short at the end of synthesis. The phosphite triesterintermediate is subjected to oxidation (the “oxidation” step) after eachcoupling reaction to yield a more stable phosphotriester intermediate.Without oxidation, the unstable phosphite triester linkage would cleaveunder the acidic conditions of subsequent synthesis steps. Letsinger etal. (1976) J. Am. Chem. Soc. 98:3655. Removal of the 5′ protecting groupof the newly added monomer (the “deprotection” step) is typicallyaccomplished by reaction with acidic solution to yield a free 5′hydroxyl group, which can be coupled to the next protected nucleosidephosphoramidite. This process is repeated for each monomer added untilthe desired sequence is synthesized.

[0021] According to some protocols, the synthesis cycle of couple, cap,oxidize, and deprotect is shortened by omitting the capping step or bytaking the oxidation step ‘outside’ of the cycle and performing a singleoxidation reaction on the completed chain. For example, oligonucleotidesynthesis according to H-phosphonate protocols will permit a singleoxidation step at the conclusion of the synthesis cycles. However,coupling yields are less efficient than those for phosphoramiditechemistry and oxidation requires longer times and harsher reagents thanamidite chemistry.

[0022] Conventional synthesis protocols of oligonucleotides are notwithout disadvantages. For example, cleavage of the DMT protecting groupunder acidic conditions gives rise to the resonance-stabilized andlong-lived bis(p-anisyl)phenylmethyl carbocation. Gilham et al. (1959)J. Am. Chem. Soc. 81:4647. Protection and deprotection of hydroxylgroups with DMT are thus readily reversible reactions, resulting in sidereactions during oligonucleotide synthesis and a lower yield than mightotherwise be obtained. To circumvent such problems, large excesses ofacid are used with DMT to achieve quantitative deprotection. As bedvolume of the polymer is increased in larger scale synthesis,increasingly greater quantities of acid are required. The acid-catalyzeddepurination which occurs during the synthesis ofoligodeoxyribonucleotides is thus increased by the scale of synthesis.Caruthers et al., in Genetic Engineering: Principles and Methods, J. K.Setlow et al., Eds. (New York: Plenum Press, 1982). Solvent use inlarger scale synthesis becomes increasingly prohibitive as well, as morewashing is required. In particular, the reagents used in the couplingstep typically are highly susceptible to hydrolysis, which requires drysolvents, further increasing the cost of solvents.

[0023] Salts that are fluid at room temperature have been investigatedas environmentally friendly solvents. These salts have been termed ‘roomtemperature ionic liquids’ (herein simply referred to as ‘ionicliquids’) and are generally composed of a heterocyclic cation, e.g. asubstituted imidazole or pyridine, and an anion such astetrafluoroborate or hexafluorophosphate, although certain organicanions such as methylsulfate (CH₃SO₄ ⁻), among others, have beendiscovered to be effective as the anion in certain organic liquids.Ionic liquids are known to dissolve a wide range of substances, bothorganic and inorganic. Ionic liquids typically are non-corrosive, havelittle or no vapor pressure under standard conditions, and exhibit lowviscosity. More information regarding ionic liquids may be gleaned fromtwo review articles by Hussey (Hussey, C. L., Adv. Molten Salt Chem.(1983) 5:185; and Hussey, C. L., Pure Appl. Chem. (1988) 60:1763).

SUMMARY OF THE INVENTION

[0024] The invention is thus addressed to the aforementioneddeficiencies in the art, and provides a novel method for synthesizingoligonucleotides, wherein the method has numerous advantages relative toprior methods such as those discussed above. The method involves formingan internucleotide bond between a first nucleoside moeity and a secondnucleoside moiety in an environment that includes an ionic liquid.

[0025] In a preferred embodiment of the invention, the second nucleosidemoiety is immobilized to an insoluble substrate. The second nucleosidemoiety on the insoluble substrate is contacted with a solution havingthe first nucleoside moiety in a solution containing ionic liquid. Aninternucleoside bond is thus formed between the first and secondnucleoside moieties. The product of the reaction is a polynucleotidewherein the first and second nucleoside moieties have been bondedtogether.

[0026] In some embodiments, the first nucleoside moiety corresponds to anucleoside phosphoramidite monomer, as in conventional polynucleotidesynthesis as described above. The invention also encompasses theformation of an internucleoside bond between two polynucleotides oroligonucleotides, or between a polynucleotide and an oligonucleotide. Insuch case, the first nucleoside moiety corresponds to the one of thepolynucleotides or oligonucleotides, and the second polynucleotidemoiety corresponds to the polynucleotide or oligonucleotide to be joinedto the first nucleoside moiety.

[0027] In particular embodiments, the reaction is geared to producing“native” polynucleotides, i.e. substantially identical to those thatmight be isolated from nature. In other embodiments, the reaction isused to synthesize polynucleotide analogues, which may have ‘modified’(not occurring in nature) phosphodiester backbones or modified basesattached to the sugar groups in the phosphodiester backbones.

[0028] In another embodiment, after the internucleotide bond has beenformed, it is modified, e.g. by oxidation, to form the ultimatepolynucleotide product. The present invention in its broadest senseencompasses materials and methods for use in forming polynucleotides,polynucleotide intermediates, and polynucleotide analogues. Theinvention also encompasses reagents and methods for synthesis ofoligonucleotides allowing the synthesis to be conducted under a widerange of conditions and allowing for the use of a variety of protectinggroups. This wide range includes the use of co-solvents along with theionic liquid in the coupling reaction.

[0029] Additional objects, advantages, and novel features of thisinvention shall be set forth in part in the descriptions and examplesthat follow and in part will become apparent to those skilled in the artupon examination of the following specifications or may be learned bythe practice of the invention. The objects and advantages of theinvention may be realized and attained by means of the instruments,combinations, compositions and methods particularly pointed out in theappended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0030] These and other features of the invention will be understood fromthe description of representative embodiments of the method herein andthe disclosure of illustrative apparatus for carrying out the method,taken together with the Figures, wherein FIG. 1 schematicallyillustrates a prior art oligonucleotide synthesis method usingphosphoramidite monomers. The known prior art methods, including the oneillustrated, do not describe the use of ionic liquids in the couplingstep where the internucleotide bond is formed.

DETAILED DESCRIPTION

[0031] Before the invention is described in detail, it is to beunderstood that unless otherwise indicated this invention is not limitedto particular materials, reagents, reaction materials, manufacturingprocesses, or the like, as such may vary. It is also to be understoodthat the terminology used herein is for purposes of describingparticular embodiments only, and is not intended to be limiting. It isalso possible in the present invention that steps may be executed indifferent sequence where this is logically possible. However, thesequence described below is preferred.

[0032] It must be noted that, as used in the specification and theappended claims, the singular forms “a,” “an” and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “an insoluble support” includes a plurality ofinsoluble supports. In this specification and in the claims that follow,reference will be made to a number of terms that shall be defined tohave the following meanings unless a contrary intention is apparent:

[0033] As used herein, polynucleotides include single or multiplestranded configurations, where one or more of the strands may or may notbe completely aligned with another. The terms “polynucleotide” and“oligonucleotide” shall be generic to polydeoxynucleotides (containing2-deoxy-D-ribose), to polyribonucleotides (containing D-ribose), to anyother type of polynucleotide which is an N-glycoside of a purine orpyrimidine base, and to other polymers in which the conventionalbackbone has been replaced with a non-naturally occurring or syntheticbackbone, e.g with a modified sugar group, or in which one or more ofthe conventional bases has been replaced with a non-naturally occurringor synthetic base.

[0034] A “nucleotide” refers to a sub-unit of a nucleic acid (whetherDNA or RNA or analogue thereof) which includes a phosphate group, asugar group and a nitrogen containing base, as well as analogs of suchsub-units. A “nucleoside” references a nucleic acid subunit including asugar group and a nitrogen containing base. A “nucleoside moiety” refersto a molecule having a sugar group and a nitrogen containing base (as ina nucleoside) as a portion of a larger molecule, such as in apolynucleotide, oligonucleotide, or nucleoside phosphoramidite. A“nucleotide monomer” refers to a molecule which is not incorporated in alarger oligo- or poly-nucleotide chain and which corresponds to a singlenucleotide sub-unit; nucleotide monomers may also have activating orprotecting groups, if such groups are necessary for the intended use ofthe nucleotide monomer. A “polynucleotide intermediate” references amolecule occurring between steps in chemical synthesis of apolynucleotide, where the polynucleotide intermediate is subjected tofurther reactions to get the intended final product, e.g. a phosphiteintermediate which isoxidized to a phosphate in a later step in thesynthesis, or a protected polynucleotide which is then deprotected. An“oligonucleotide” generally refers to a nucleotide multimer of about 2to 100 nucleotides in length, while a “polynucleotide” includes anucleotide multimer having any number of nucleotides. It will beappreciated that, as used herein, the terms “nucleoside” and“nucleotide” will include those moieties which contain not only thenaturally occurring purine and pyrimidine bases, e.g., adenine (A),thymine (T), cytosine (C), guanine (G), or uracil (U), but also modifiedpurine and pyrimidine bases and other heterocyclic bases which have beenmodified (these moieties are sometimes referred to herein, collectively,as “purine and pyrimidine bases and analogs thereof”). Suchmodifications include, e.g., methylated purines or pyrimidines, acylatedpurines or pyrimidines, and the like, or the addition of a protectinggroup such as acetyl, difluoroacetyl, trifluoroacetyl, isobutyryl,benzoyl, or the like. The purine or pyrimidine base may also be ananalog of the foregoing; suitable analogs will be known to those skilledin the art and are described in the pertinent texts and literature.Common analogs include, but are not limited to, 1-methyladenine,2-methyladenine, N6-methyladenine, N6-isopentyladenine,2-methylthio-N6-isopentyladenine, N,N-dimethyladenine, 8-bromoadenine,2-thiocytosine, 3-methylcytosine, 5-methylcytosine, 5-ethylcytosine,4-acetylcytosine, 1-methylguanine, 2-methylguanine, 7-methylguanine,2,2-dimethylguanine, 8-bromoguanine, 8-chloroguanine, 8-aminoguanine,8-methylguanine, 8-thioguanine, 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, 5-ethyluracil, 5-propyluracil,5-methoxyuracil, 5-hydroxymethyluracil, 5-(carboxyhydroxymethyl)uracil,5-(methylaminomethyl)uracil, 5-(carboxymethylaminomethyl)-uracil,2-thiouracil, 5-methyl-2-thiouracil, 5-(2-bromovinyl)uracil,uracil-5-oxyacetic acid, uracil-5-oxyacetic acid methyl ester,pseudouracil, 1-methylpseudouracil, queosine, inosine, 1-methylinosine,hypoxanthine, xanthine, 2-aminopurine, 6-hydroxyaminopurine,6-thiopurine and 2,6-diaminopurine.

[0035] An “internucleotide bond” refers to a chemical linkage betweentwo nucleoside moieties, such as a phosphodiester linkage in nucleicacids found in nature, or such as linkages well known from the art ofsynthesis of nucleic acids and nucleic acid analogues. Aninternucleotide bond may comprise a phospho or phosphite group, and mayinclude linkages where one or more oxygen atoms of the phospho orphosphite group are either modified with a substituent or replaced withanother atom, e.g. a sulfur atom, or the nitrogen atom of a mono- ordi-alkyl amino group.

[0036] A “group” includes both substituted and unsubstituted forms.Typical substituents include one or more lower alkyl, any halogen,hydroxy, or aryl, or optionally substituted on one or more availablecarbon atoms with a nonhydrocarbyl substituent such as cyano, nitro,halogen, hydroxyl, or the like. Any substituents are typically chosen soas not to substantially adversely affect reaction yield (for example,not lower it by more than 20% (or 10%, or 5% or 1%) of the yieldotherwise obtained without a particular substituent or substituentcombination). An “acetic acid” includes substituted acetic acids such asdi-chloroacetic acid (DCA) or tri-chloroacetic acid (TCA).

[0037] A “phospho” group includes a phosphodiester, phosphotriester, andH-phosphonate groups. In the case of either a phospho or phosphitegroup, a chemical moiety other than a substituted 5-membered furyl ringmay be attached to 0 of the phospho or phosphite group which linksbetween the furyl ring and the P atom.

[0038] A “protecting group” is used in the conventional chemical senseto reference a group which reversibly renders unreactive a functionalgroup under specified conditions of a desired reaction. After thedesired reaction, protecting groups may be removed to deprotect theprotected functional group. All protecting groups should be removable(and hence, labile) under conditions which do not degrade a substantialproportion of the molecules being synthesized. In contrast to aprotecting group, a “capping group” permanently binds to a segment of amolecule to prevent any further chemical transformation of that segment.A “hydroxyl protecting group” refers to a protecting group where theprotected group is a hydroxyl. A “reactive-site hydroxyl” is theterminal 5′-hydroxyl during 3′-5′ polynucleotide synthesis and is the3′-hydroxyl during 5′-3′ polynucleotide synthesis. An “acid labileprotected hydroxyl” is a hydroxyl group protected by a protecting groupthat can be removed by acidic conditions. Similarly, an “acid labileprotecting group” is a protecting group that can be removed by acidicconditions. Preferred protecting groups that are capable of removalunder acidic conditions (“acid-labile protecting groups”) include thosesuch as tetrahydropyranyl groups, e.g. tetrahydropyran-2-yl and4-methoxytetrahydropyran-2-yl; an arylmethyl group with n aryl groups(where n=1 to 3) and 3-n alkyl groups such as an optionally substitutedtrityl group, for example a monomethoxytrityl for oligoribonucleotidesynthesis and a dimethoxytrityl for oligodeoxyribonucleotide synthesis,pixyl; isobutyloxycarbonyl; t-butyl; and dimethylsilyl. A trityl groupis a triphenylmethyl group. Suitable protecting groups are described in“Protective Groups in Organic Synthesis” by T. W. Green, WileyInterscience.

[0039] The term “alkyl” as used herein, unless otherwise specified,refers to a saturated straight chain, branched or cyclic hydrocarbongroup of 1 to 24, typically 1-12, carbon atoms, such as methyl, ethyl,n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, cyclopentyl,isopentyl, neopentyl, hexyl, isohexyl, cyclohexyl, 3-methylpentyl,2,2-dimethylbutyl, and 2,3-dimethylbutyl. The term “lower alkyl” intendsan alkyl group of one to eight carbon atoms, and includes, for example,methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl,cyclopentyl, isopentyl, neopentyl, hexyl, isohexyl, cyclohexyl,3-methylpentyl, 2,2-dimethylbutyl, and 2,3-dimethylbutyl. The term“cycloalkyl” refers to cyclic alkyl groups such as cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.

[0040] The term “alkenyl” as used herein, unless otherwise specified,refers to a branched, unbranched or cyclic (in the case of C5 and C6)hydrocarbon group of 2 to 24, typically 2 to 12, carbon atoms containingat least one double bond, such as ethenyl, vinyl, allyl, octenyl,decenyl, and the like. The term “lower alkenyl” intends an alkenyl groupof two to eight carbon atoms, and specifically includes vinyl and allyl.The term “cycloalkenyl” refers to cyclic alkenyl groups.

[0041] The term “alkynyl” as used herein, unless otherwise specified,refers to a branched or unbranched hydrocarbon group of 2 to 24,typically 2 to 12, carbon atoms containing at least one triple bond,such as acetylenyl, ethynyl, n-propynyl, isopropynyl, n-butynyl,isobutynyl, t-butynyl, octynyl, decynyl and the like. The term “loweralkynyl” intends an alkynyl group of two to eight carbon atoms, andincludes, for example, acetylenyl and propynyl, and the term“cycloalkynyl” refers to cyclic alkynyl groups.

[0042] The term “aryl” as used herein refers to an aromatic speciescontaining 1 to 5 aromatic rings, either fused or linked, and eitherunsubstituted or substituted with 1 or more substituents typicallyselected from the group consisting of amino, halogen and lower alkyl.Preferred aryl substituents contain 1 to 3 fused aromatic rings, andparticularly preferred aryl substituents contain 1 aromatic ring or 2fused aromatic rings. Aromatic groups herein may or may not beheterocyclic. The term “aralkyl” intends a moiety containing both alkyland aryl species, typically containing less than about 24 carbon atoms,and more typically less than about 12 carbon atoms in the alkyl segmentof the moiety, and typically containing 1 to 5 aromatic rings. The term“aralkyl” will usually be used to refer to aryl-substituted alkylgroups. The term “aralkylene” will be used in a similar manner to referto moieties containing both alkylene and aryl species, typicallycontaining less than about 24 carbon atoms in the alkylene portion and 1to 5 aromatic rings in the aryl portion, and typically aryl-substitutedalkylene. Exemplary aralkyl groups have the structure —(CH2)j-Ar whereinj is an integer in the range of 1 to 24, more typically 1 to 6, and Aris a monocyclic aryl moiety.

EXAMPLES

[0043] The practice of the present invention will employ, unlessotherwise indicated, conventional techniques of synthetic organicchemistry, biochemistry, molecular biology, and the like, which arewithin the skill of the art. Such techniques are explained fully in theliterature.

[0044] The following examples are put forth so as to provide those ofordinary skill in the art with a complete disclosure and description ofhow to prepare and use the compounds disclosed and claimed herein.Efforts have been made to ensure accuracy with respect to numbers (e.g.,amounts, temperature, etc.) but some errors and deviations should beaccounted for. Unless indicated otherwise, parts are parts by weight,temperature is in ° C. and pressure is at or near atmospheric. Standardtemperature and pressure are defined as 20° C. and 1 atmosphere.

[0045] The synthesis of polynucleotides has been well-studied, andmethods incorporate both aqueous and organic solvents. It is well knownthat changing the solvent in a reaction system frequently affects theperformance of the reaction, sometimes profoundly. The ionic nature ofionic liquids fundamentally differs from molecular nature of aqueous ororganic solvents used in various steps of the polynucleotide synthesiscycle. Potential problems include changes of chemical mechanism,possibly favoring different products due to the ionic nature of thesolvent. Stabilization of charged reaction intermediates due tointeraction with the ionic liquid, or chemical reaction with componentsof the ionic liquid itself might be expected. Will the short-livedreaction intermediates found in conventional solvents be long-livedstable intermediates in an ionic liquid? Will changes in the relativestabilities of reaction intermediates change the available reactionpathways? Will the expected changes in reaction kinetics shift thebalance between thermodynamic and kinetic control, and hence producedifferent products?

[0046] We determined to study the effect of the coupling reaction inionic liquid solvent as an alternative to molecular solvents (aqueousand organic solvents). We have discovered that, despite the previouslymentioned potential problems, we were able to achieve coupling ofnucleoside moieties via formation of an internucleotide bond in ionicliquids. We have now found that various advantages exist in performingthe coupling reaction in ionic liquids. One advantage we found was thatthe hydrophobicity of ionic liquid led to reduced problems in dealingwith hydrolysis of the reactants due to water in the reactionenvironment. Less solvent may be used to wash in between coupling steps,and ionic liquid solvents may be recovered more easily, when compared toprior art methods. This may be particularly useful in large-scalesynthesis, where lots of washing and solvents are required.

[0047] Particularly useful phosphoramidites, their preparation, andtheir use are described in detail in U.S. Pat. No. 5,902,878; U.S. Pat.No. 5,700,919; U.S. Pat. No. 4,668,777; U.S. Pat. No. 4,415,732; PCTpublication WO 98/41531 and the references cited therein, among others.

[0048] The chemical synthesis of thymidine-thymidylate dimers in ionicliquid were preformed by the following protocol:

[0049] 3 Å molecular sieves were activated by drying in a vacuum oven at200° C. overnight. A small number of sieves were placed in a 5 ml, roundbottom flask with a 14/20 ground glass joint that was then sealed with arubber septum. 3 ml of 1-ethyl-3-methyl-1H-imidazoliumtrifluoromethanesulfonate (Aldrich Chemical Company, Milwaukee, Wis.USA) was added to the flask and the liquid allowed to dry overnight.5′-Dimethoxytrityl-2′-deoxythymidine,3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 0.38 grams, 0.5mmol, was added to the flask and the solution shaken until the reagenthad dissolved. A small amount of the solution was removed from the flaskand placed in an NMR tube for analysis by ³¹P NMR using an externallock. The resulting NMR spectrum showed the presence of the startingmaterial nucleoside phosphoramidite at δ 147.18 ppm relative tophosphoric acid.

[0050] 3′-Acetyl Thymidine (ChemGenes Corp., Waltham Mass. USA) 0.14grams, 0.5 mmol was added to the mixture along with tetrazole 0.18grams, 2.5 mmol. The solution was shaken on a wrist action shaker untilthe reagents were completely dissolved. An aliquot of the reactionmixture was removed from the flask and placed in an NMR tube foranalysis by ³¹P NMR using an external lock. The resulting NMR spectrumshowed complete conversion of the starting material nucleosidephosphoramidite at δ 147.18 ppm to the phosphite triester at δ 139.16ppm.

[0051] In another example, 3 A molecular sieves were activated by dryingin a vacuum oven at 200° C. overnight. A small number of sieves wereplaced in a 5 ml, round bottom flask with a 14/20 ground glass jointthat was then sealed with a rubber septum. In this example, 3 ml of1-butyl-3-methyl-imidazolium tetrafluoroborate (Solvent Innovation GmbH,50679 Köln, Germany) was added to the flask and the liquid allowed todry overnight. 5′-Dimethoxytrityl-2′-deoxyThymidine,3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 0.38 grams, 0.5mmol, was added to the flask and the solution shaken until the reagenthad dissolved. A small amount of the solution was removed from the flaskand placed in an NMR tube for analysis by ³¹P NMR using an externallock. The resulting NMR spectrum showed the presence of the startingmaterial nucleoside phosphoramidite at δ 146.94 ppm relative tophosphoric acid.

[0052] 3′-Acetyl Thymidine (ChemGenes Corp., Waltham Mass.) 0.14 grams,0.5 mmol was added to the mixture along with tetrazole 0.18 grams, 2.5mmol. The solution was shaken on a wrist action shaker until thereagents were completely dissolved. An aliquot of the reaction mixturewas removed from the flask and placed in an NMR tube for analysis by ³¹PNMR using an external lock. The resulting NMR spectrum showed completeconversion of the starting material nucleoside phosphoramidite at δ146.94 ppm to the phosphite triester diastereomers centered at δ 138.98ppm.

[0053] In general, the product of the coupling reaction, when performedin a solid phase system, may be represented by the following structuralformula:

[0054] Wherein:

[0055] ◯ represents the solid support or a support-bound oligonucleotidechain;

[0056] A represents H or an optionally protected hydroxyl group;

[0057] B is a purine or pyrimidine base whose exocyclic amine functionalgroup is optionally protected; and

[0058] R is a suitable protecting group,

[0059] “Y” is hydrido or hydrocarbyl, typically alkyl, alkenyl, aryl,aralkyl, or cycloalkyl. Preferably, Y represents: lower alkyl;electron-withdrawing β-substituted aliphatic, particularlyelectron-withdrawing β-substituted ethyl such as β-trihalomethyl ethyl,β-cyanoethyl, β-sulfoethyl, β-nitro-substituted ethyl, and the like;electron-withdrawing substituted phenyl, particularly halo-, sulfo-,cyano- or nitro-substituted phenyl; or electron-withdrawing substitutedphenylethyl. Most preferably, Y represents methyl, β-cyanoethyl, or4-nitrophenylethyl.

[0060] In this formula, the sugar and the base to the 5′ side of thephosphorus atom (P) corresponds to one nucleoside moiety, and the sugarand the base to the 3′ side of the phosphorus atom (P) correspond to theother nucleoside moiety.

[0061] Ionic liquids that may be used include organic salts that arefluid below about 80° C. at around normal atmospheric pressure (about 1atmosphere at sea level). The organic salts generally have an organiccation and either an inorganic or organic counterion. The organic cationis preferably an N-substituted pyridine having the following structure:

[0062] wherein R is alkyl and each R′ is independently selected fromhyrido, alkyl, or halogen;

[0063] or a 1,3 di-substituted imidazole having the following structure:

[0064] wherein each R is independently selected from alkyl, each R′ isindependently selected from hydrido, alkyl, or halogen, and R″ isselected from hydrido or methyl.

[0065] Preferred organic cations include 1,3-dimethyl-imidazolium,1-ethyl-3-methyl-imidazolium, 1-butyl-3-methyl-imidazolium,1-hexyl-3-methyl-imidazolium, 1-decyl-3-methyl-imidazolium,1-dodecyl-3-methyl-imidazolium, 1-methyl-3-octyl-imidazolium,1-methyl-3-tetradecyl-imidazolium, 1,2-dimethyl-3-propyl-imidazolium,1-ethyl-2,3-dimethyl-imidazolium, 1-butyl-2,3-dimethyl-imidazolium,N-ethylpyridinium, N-butylpyridinium, N-hexylpyridinium,4-methyl-N-butyl-pyridinium, 3-methyl-N-butyl-pyridinium,4-methyl-N-hexyl-pyridinium, 3-methyl-N-hexyl-pyridinium,4-methyl-N-octyl-pyridinium, 3-methyl-N-octyl-pyridinium,3,4-dimethyl-N-butyl-pyridinium, and 3,5-dimethyl-N-butyl-pyridinium.

[0066] Preferred anions of the ionic liquid are chloride (Cl⁻), bromide(Br⁻), tetrafluoroborate ([BF₄]⁻), hexafluorophosphate ([PF₆]⁻),[SbF₆]⁻, [CuCl₂]⁻, [AlCl₄]⁻, [Al₂Cl₇]⁻, [Al₃Cl₁₀]⁻, methylsulfate(CH₃SO₄ ⁻), trifluoroacetate (CF₃CO₂ ⁻), heptafluorobutanoate(CF₃(CF₂)₂CO₂ ⁻), triflate (CF₃SO₂ ⁻), nonaflate (C₂F₅SO₂ ⁻),bis(trifluoromethylsulfonyl)imide ((CF₃SO₂)₂N⁻),bis(perfluoroethylsulfonyl)imide ((C₂F₅SO₂)₂N⁻), andtris(trifluoromethylsulfonyl)methide ((CF₃SO₂)₃C⁻). Ionic liquids areavailable from Covalent Associates (Woburn, Mass.), Solvent Innovation(Koln, Germany), Aldrich Chemical Company (Milwaukee, Wis.), and AcrosOrganics (Geel, Belgium).

[0067] In one embodiment, to perform the coupling reaction, at least oneof the chemical species having a nucleoside moiety is dissolved in asolution having at least 98 percent by weight of ionic liquid, whereuponthe other chemical specie having a nucleoside moiety (the secondnucleoside moiety) is contacted with the solution containing ionicliquid and the first nucleoside moiety. In other embodiments, thesolution has at least about 90% ionic liquid, or at least about 75%ionic liquid, or at least about 50% ionic liquid, or at least about 25%ionic liquid, or at least about 10% ionic liquid. Co-solvents that maybe mixed into the ionic liquid include but are not limited toacetonitrile, tetrahydrofuran, dimethylformamide, methylene chloride,propylene carbonate, adiponitrile, toluene, dioxane, dimethylsulfoxide,and N-methyl pyrrolidone. An activator compound is typically included ina concentration of about 0.05 molar up to about 0.5 molar. The activatoris generally tetrazole, S-ethyl-thiotetrazole, 4-nitrotriazole, ordicyanoimidazole, although other acidic azoles may be used. Onepotential advantage of using an ionic liquid is that the ionic liquidmay serve as the activator.

[0068] In the conventional synthesis method depicted schematically inFIG. 1, it is typical to use an aqueous solution of iodine for theoxidation step. However, phosphoramidite reagents that have beenactivated for coupling are highly reactive with water. The invention maybe extended to include using ionic liquids as solvents elsewhere in thesynthesis cycle to reduce or substantially eliminate the presence ofwater during oxidation and deprotection.

[0069] In one embodiment of the invention, the first nucleoside moietyis a monomer nucleoside phosphoramidite, which is coupled to a freehydroxyl of a second nucleoside moiety, analogous to conventionalpolynucleotide synthesis. The invention also encompasses the formationof an internucleoside bond between two polynucleotides oroligonucleotides, or between a polynucleotide and an oligonucleotide. Insuch case, the first nucleoside moiety corresponds to the one of thepolynucleotides or oligonucleotides, and the second polynucleotidemoiety corresponds to the polynucleotide or oligonucleotide to be joinedto the first nucleoside moiety. The skilled practitioner in the art willrealize that one of the nucleoside moieties must be activated, as in aphosphoramidite. Such modification is well known in the art.

[0070] As explained earlier herein, the method of the invention alsolends itself to synthesis of polynucleotides in the 5′-to-3′ direction.In such a case, the initial step of the synthetic process involvesattachment of an initial nucleoside to a solid support at the 5′position, leaving the 3′ position available for covalent binding of asubsequent monomer. The coupling reaction in which the nucleosidemonomer becomes covalently attached to the 3′ hydroxyl moiety of thesupport bound nucleoside is conducted under reaction conditionsidentical to those described for the 3′-to-5′ synthesis. The synthesiscycle is then continued with the (optional) capping step, the oxidationof the internucleotide bond, and the deprotection of the active sitehydroxyl in preparation for the next synthesis cycle, which is repeateduntil a polynucleotide having the desired sequence and length isobtained. Following synthesis, the polynucleotide may, if desired, becleaved from the solid support. The details of the synthesis in the5′-to-3′ direction will be readily apparent to the skilled practitionerbased on the prior art and the disclosure contained herein.

[0071] The coupling reaction as described herein may easily be adaptedto be performed in a conventional automated oligonucleotide synthesizerutilizing an insoluble substrate to immobilize the polynucleotidesduring synthesis. Such methodology will be apparent to those skilled inthe art and is described in the pertinent texts and literature, e.g., inD.M. Matteuci et al. (1980) Tet. Lett. 521:719 and U.S. Pat. No.4,500,707. Examples of suitable support materials include, but are notlimited to, polysaccharides such as agarose (e.g., that availablecommercially as Sepharose(g, from Pharmacia) and dextran (e.g., thoseavailable commercially under the tradenames Sephadex® and Sephacyl®,also from Pharmacia)

[0072] While the foregoing embodiments of the invention have been setforth in considerable detail for the purpose of making a completedisclosure of the invention, it will be apparent to those of skill inthe art that numerous changes may be made in such details withoutdeparting from the spirit and the principles of the invention.Accordingly, the invention should be limited only by the followingclaims.

[0073] All patents, patent applications, and publications mentionedherein are hereby incorporated by reference in their entireties.

What is claimed is:
 1. A method of forming an internucleotide bondcomprising contacting a free hydroxyl of a growing polynucleotide with asolution comprising at least about ten percent by weight of an ionicliquid and a nucleotide monomer.
 2. The method of claim 1 wherein theionic liquid is an organic salt comprising a substituted heterocyclicorganic cation.
 3. The method of claim 2 wherein the organic saltfurther comprises an anion selected from chloride (Cl⁻), bromide (Br⁻),tetrafluoroborate ([BF₄]⁻), hexafluorophosphate ([PF₆]⁻), [SbF₆]⁻,[CuCl₂]⁻, [AlCl₄]⁻, [Al₂Cl₇]⁻, [Al₃Cl₁₀]⁻, methylsulfate (CH₃SO₄ ⁻),trifluoroacetate (CF₃CO₂ ⁻), heptafluorobutanoate (CF₃(CF₂)₂CO₂ ⁻),triflate (CF₃SO₂ ⁻), nonaflate (C₂F₅SO₂ ⁻),bis(trifluoromethylsulfonyl)imide ((CF₃SO₂)₂N⁻),bis(perfluoroethylsulfonyl)imide ((C₂F₅SO₂)₂N⁻), andtris(trifluoromethylsulfonyl)methide ((CF₃SO₂)₃C⁻).
 4. The method ofclaim 2 wherein the organic salt is characterized as being a liquid whenbeing >98% pure and at standard temperature and pressure.
 5. The methodof claim 2 wherein the cation is an N-substituted pyridine having theformula

wherein R is alkyl and each R′ is independently selected from hyrido,alkyl, or halogen group.
 6. The method of claim 2 wherein the cation hasthe formula

wherein each R is independently selected from alkyl, each R′ isindependently selected from hydrido, alkyl, or halogen, and R” isselected from hydrido or methyl.
 7. The method of claim 1 wherein theionic liquid is an organic salt comprising a cation selected from anN-substituted pyridine and a 1,3-disubstituted imidazole.
 8. A method offorming an internucleotide bond between a first nucleoside moiety and asecond nucleoside moiety, the method comprising (a) dissolving the firstnucleoside moiety in a solution comprising at least ten percent byweight of an ionic liquid, and (b) contacting the second nucleosidemoiety with the product of (a) to form the internucleotide bond.
 9. Themethod of claim 8, further comprising (c) contacting the internucleotidebond with an oxidizing reagent to oxidize the internucleotide bond. 10.The method of claim 8 wherein the second nucleoside moiety isimmobilized on a solid support.
 11. The method of claim 8 wherein theionic liquid is an organic salt comprising a substituted heterocyclicorganic cation.
 12. The method of claim 11 wherein the organic saltfurther comprises an anion selected from chloride (Cl⁻), bromide (Br⁻),tetrafluoroborate ([BF₄]⁻), hexafluorophosphate ([PF₆]⁻), [SbF₆]⁻,[CuCl₂]⁻, [AlCl₄]⁻, [Al₂Cl₇]⁻, [Al₃Cl₁₀]⁻, methylsulfate (CH₃SO₄ ⁻),trifluoroacetate (CF₃CO₂ ⁻), heptafluorobutanoate (CF₃(CF₂)₂CO₂ ⁻),triflate (CF₃SO₂ ⁻), nonaflate (C₂F₅SO₂ ⁻),bis(trifluoromethylsulfonyl)imide ((CF₃SO₂)₂N⁻),bis(perfluoroethylsulfonyl)imide ((C₂F₅SO₂)₂N⁻), andtris(trifluoromethylsulfonyl)methide ((CF₃SO₂)₃C⁻).
 13. The method ofclaim 1 1 wherein the organic salt is characterized as being a liquidwhen being >98% pure and at standard temperature and pressure.
 14. Themethod of claim 11 wherein the cation is an N-substituted pyridinehaving the formula

wherein R is alkyl and each R′ is independently selected from hyrido,alkyl, or halogen group.
 15. The method of claim 11 wherein the cationhas the formula

wherein each R is independently selected from alkyl, each R′ isindependently selected from hydrido, alkyl, or halogen, and R″ isselected from hydrido or methyl.
 16. The method of claim 8 wherein theionic liquid is an organic salt comprising a cation selected from anN-substituted pyridine and a 1,3-disubstituted imidazole.